Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene – Relevance for toxicological assessment
Introduction
Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) was the first insect growth regulator approved in the 1970’s by the Environmental Protection Agency (EPA, USA) after extensive studies showing low toxicity to vertebrates (Zoecon Corporation, 1974). Insect growth regulators typically perturb enzymatic and hormonally regulated processes that are relatively specific to insect physiology (Retnakaran et al., 1985, Dhadialla et al., 1998). Methoprene is one of the most widely used and successful insect growth regulators, exerting its toxicity to target insects by acting as a juvenile hormone agonist (Dhadialla et al., 1998), preventing the emergence of progeny without killing the adult insects (Amos and Williams, 1977). Although these designed pesticides usually show low toxicity to non-target organisms, due to the absence of enzymatic or hormone-regulated processes that these insecticides disrupt, deleterious effects, specially developmental abnormalities, have been already reported for methoprene. Non-target aquatic insects (Miura and Takahashi, 1973, Breaud et al., 1977), crustaceans (Breaud et al., 1977, Templeton and Laufer, 1983) and, at higher concentrations, some fish species (Quistad et al., 1976, McKague and Pridmore, 1978) were observed to be affected by the presence of the insecticide. Despite the large number of ecotoxicity studies available, the mechanisms underlying the toxicity of methoprene on such non-target species have been scarcely addressed.
The interference of other pesticides with basic mitochondrial bioenergetic parameters has been clearly established for such compounds as DDT (Moreno and Madeira, 1991) and DDE (Ferreira et al., 1997), dinoseb (Palmeira et al., 1994), paraquat (Palmeira et al., 1995), endosulfan (da Silva et al., 1998), and ethylazinphos (Videira et al., 2001) among others. The hypothesis of methoprene influencing mitochondrial bioenergetics is further supported by the fact that this insecticide has been shown to impair the redox flow through the enzymatic complexes of the respiratory system of Bacillus stearothermophilus (Monteiro et al., 2005), a thermophilic eubacterium extensively used as a model for toxicological evaluation of environmental pollutants (Donato et al., 2000, Martins et al., 2003, Monteiro et al., 2005). These facts motivated us to perform evaluation studies on the putative toxicity of methoprene as exerted over mitochondrial physiologic features.
Mitochondria have proved to be valid models for studying cell toxicity of many xenobiotics, the data obtained from mitochondrial studies being generally correlated with cytotoxicity parameters reported in cell cultures and whole organisms (Blondin et al., 1987, Knobeloch et al., 1990, Argese et al., 1995). Additionally, the toxicological relevance of mitochondrial studies is intimately related to the fact that most of the energy involved in cellular metabolism and intermediary metabolic compounds are generated at the expense of mitochondrial respiration.
Another mitochondrial specific physiological process, the mitochondrial permeability transition (MPT), which has been implicated as a control point for cell death, being caused by the opening of pores in the mitochondrial membranes, has also been studied as a target for methoprene action on mitochondria. The permeability transition pore (PTP) is a high-conductance nonspecific pore presumably formed by a supramolecular complex spanning the mitochondrial double-membrane system mainly at contact sites. The core of the PTP is thought to involve the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, the adenine nucleotide translocator (ANT) in the inner membrane and cyclophilin D in the mitochondrial matrix (Crompton, 1999). The MPT results in several consequences for mitochondria including an increase in mitochondrial matrix volume (Kaasik et al., 2007) that can facilitate the release of apoptotic mediators such as cytochrome c from mitochondria (Zamzami et al., 1996, Hirsch et al., 1998, Skulachev, 2000), the loss of mitochondrial membrane potential (ΔΨ) and oxidative phosphorylation uncoupling (Zoratti and Szabo, 1995).
In the present work we carried out a systematic study on the influence of methoprene on mitochondrial energetic function and on the induction of the mitochondrial permeability transition pore, attempting to provide relevant hints about the possible action mechanisms of the insecticide which may account for its toxicity manifestations on non-target species, including noxious effects on vertebrates.
Section snippets
Chemicals
The insecticide methoprene (94.9% purity) was obtained from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade for research. Equal volumes of ethanol solvent added to controls (up to 3 μl) were without effect on measured parameters.
Isolation of rat liver mitochondria
Mitochondria were isolated from livers of male Wistar rats (3 months old) by conventional methods (Gazotti et al., 1979) with slight modifications. Homogenization medium contained 250 mM sucrose, 10 mM HEPES (pH 7.4), 0.5 mM EGTA, and 0.1%
Results
The effect of methoprene on the mitochondrial membrane potential (ΔΨ) was studied by following the fluctuations associated with mitochondrial oxidative phosphorylation (Fig. 1; Table 1). The results were different according to which respiratory substrate was used (succinate or ascorbate + TMPD). Initially, mitochondria developed a ΔΨ between 210 and 220 mV (negative inside) upon substrate addition. Afterwards, the incubation with increasing concentrations of methoprene (from 40 to 100 nmol mg−1 of
Discussion
Toxic effects of methoprene on non-target organisms have been reported, especially on immature crustaceans and fish (Antunes-Kennion and Kennedy, 2001). Some studies suggest methoprene interference with endocrine pathways of immature forms of crustaceans (Harmon et al., 1995) and anurans (Olmstead and LeBlanc, 2001). However, insecticide toxicity on non-target organisms is far from being fully clarified. Due to its hydrophobic nature, methoprene is liable for membrane interference and
References (60)
- et al.
Mitochondrial cytochrome c: preparation and activity of native and chemically modified cytochrome c
Meth. Enzymol.
(1978) - et al.
Cyclosporin A-sensitive and insensitive mechanisms produce the permeability transition in mitochondria
Biochem. Biophys. Res. Commun.
(1989) - et al.
Tamoxifen inhibits induction of the mitochondrial permeability transition by Ca2+ and inorganic phosphate
Toxicol. Appl. Pharmacol.
(1998) Mitochondrial respiratory control and the polarographic measurements of ADP/O ratios
Meth. Enzymol.
(1967)- et al.
Interactions of 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene with mitochondrial oxidative phosphorylation
Biochem. Pharmacol.
(1997) - et al.
Cyclosporin A inhibits thyroid hormone-induced shortening of the tadpole tail through membrane permeability transition
Comp. Biochem. Physiol. B: Biochem. Mol. Biol.
(2003) - et al.
From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state
Biochim. Biophys. Acta
(1998) The mitochondrial permeability transition: from biochemical curiosity to pathophysiological mechanisms
Gastroenterology
(1998)- et al.
Activation energies of the ATPase activity of sarcoplasmatic reticulum
Biochim. Biophys. Res. Comm.
(1974) - et al.
Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan
Toxicol. In Vitro
(2003)
Toxicity of methoprene as assessed by the use of a model microorganism
Toxicol. In Vitro
Interference of parathion with mitochondrial bioenergetics
Biochim. Biophys. Acta
Mitochondrial bioenergetics as affected by DDT
Biochim. Biophys. Acta
Inhibition of mitochondrial bioenergetics by carbaryl is only evident for higher concentrations – relevance for carbaryl toxicity mechanisms
Chemosphere
Mechanisms of energy transduction
Interactions of herbicides 2,4-d and dinoseb with liver mitochondrial bioernergetics
Toxicol. Appl. Pharmacol.
Mitochondrial bioenergetics as affected by the herbicide paraquat
Biochim. Biophys. Acta
Benzoquinone inhibits the voltage-dependent induction of the mitochondrial permeability transition caused by redox-cycling naphthoquinones
Toxicol. Appl. Pharmacol.
Cholesterol and bile acids via acetate from the insect juvenile hormone analog methoprene
Life Sci.
Environmental degradation of the insect growth regulator methoprene. IX. Metabolism by bluegill fish
Pestic. Biochem. Physiol.
Control of mitochondrial respiration
FEBS Lett.
Preparation and properties of succinic-cytochrome c reductase (complexes II–III)
Meth. Enzymol.
Ethylazinphos interaction with membrane lipid organization induces increase of proton permeability and impairment of mitochondrial bioenergetic functions
Toxicol. Appl. Pharmacol.
Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis
FEBS Lett.
The mitochondrial permeability transition
Biochem. Biohys. Acta
Insect growth regulators: some effects of methoprene and hydroprene on productivity of several stored grain insects
Aust. J. Zool.
Membrane partition of organophosphorus and organochlorine insecticides and its implications for mechanisms of toxicity
Pest. Sci.
Submitochondrial particles as toxicity biosensors of chlorophenols
Environ. Toxicol. Chem.
Mammalian mitochondria as in vitro monitors of water quality
Bull. Environ. Contam. Toxicol.
Cited by (17)
Chitosan quaternary ammonium salt induced mitochondrial membrane permeability transition pore opening study in a spectroscopic perspective
2020, International Journal of Biological MacromoleculesCitation Excerpt :Centrifugation for the extraction of mitochondria was run on a TGM-20 high-speed freezing centrifuge (Xiangyi, Changsha, China). Based on the different density of each component in cells, mitochondria were extracted by differential centrifugation in this experiment [21]. Rat liver samples were quickly removed and placed in a beaker, washed in ice-cold homogenization buffer: solution A (220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 2 mM Tris-HCl and 0.1 mM EDTA, pH = 7.4).
Microcalorimetric and microscopic studies of the effect of chitosan quaternary ammonium salt on mitochondria
2019, International Journal of Biological MacromoleculesCitation Excerpt :Centrifugations for the extraction of mitochondria were run on a TGM-20 high-speed freezing centrifuge (Xiangyi, Changsha, China). Based on the difference of density of each component in cells, mitochondria were extracted by differential centrifugation in this experiment [19]. Rat liver samples were quickly removed and placed in a beaker, washed in ice-cold homogenization buffer: solution A (220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 2 mM Tris-HCl and 0.1 mM EDTA, pH 7.4).
Indium (III) induces isolated mitochondrial permeability transition by inhibiting proton influx and triggering oxidative stress
2017, Journal of Inorganic BiochemistryCitation Excerpt :And all the other stock solutions were prepared with sterile double-distilled water acquired from a water purification system (Simplicity, Millipore Corp, Billerica, MA). Female Wistar rat (130–150 g) was executed and the liver mitochondria were isolated with differential centrifugation method [24]. In brief, fresh liver tissue was cut into pieces and then homogenized with a set of Dounce Tissue Grinders (Wheather) [25] in Buffer A: 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 2 mM Tris-HCl and 1 mM EDTA, pH 7.4.
Mitochondrial dysfunction induced by ultra-small silver nanoclusters with a distinct toxic mechanism
2016, Journal of Hazardous MaterialsCitation Excerpt :The contents of silver element in Ag NCs solution were determined by inductively coupled plasma (ICP) spectrometry (ICPS-7500, Shimadzu, Kyoto, Japan). Rat liver mitochondria were isolated from Wistar rats (150–200 g) according to a typical method, standard differential centrifugation procedures with a little modification, of which the details were described in Section 1, Supplementary material [36]. The mitochondrial protein concentration was determined by the method described by Lowry et al., using bovine serum albumin as the standard [37].
Toxicity of polyhydroxylated fullerene to mitochondria
2016, Journal of Hazardous MaterialsToxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems
2014, Toxicology in VitroCitation Excerpt :Given the interest linuron has aroused in terms of its toxicological potential towards either the environment or the human health, the biological activity of this herbicide was assessed in the present work using two in vitro model systems: a Gram-positive bacterium, Bacillus stearothermophilus, and rat liver mitochondria. These models were chosen on the basis of data provided by previous studies in our laboratory showing that they are useful tools for a preliminary quantitative evaluation of the toxicity exerted by a wide range of xenobiotics (Donato et al., 1997a; Fernandes et al., 2008; Martins et al., 2005; Monteiro et al., 2005, 2008; Pereira et al., 2009). The growth, cell viability and respiratory activity of B. stearothermophilus have proved to provide sensitive biological parameters to assess cytotoxicity effects of chemical compounds, showing a good correlation with other bioindicators of chemical stress provided by eukaryotic organisms (Donato et al., 1997b; Monteiro et al., 2008; Pereira et al., 2009).